Hepatic encephalopathy (HE) is an important cause of morbidity and mortality in patients with severe liver failure. Acute HE associated with fulminant hepatic failure has an extremely poor prognosis and specific therapy is not available, short of an emergency liver transplantation. Although its pathogenesis remains poorly understood, ammonia is strongly implicated as a neurotoxin, and astrocytes appear to be the primary target of ammonia neurotoxicity. Additionally, altered bioenergetics and oxidative stress are thought to play a major role in this disorder. These facts led to a consideration of the involvement of mitochondrial permeability transition (MPT) as a factor in the pathogenesis of HE and ammonia neurotoxicity. The MPT is a Ca2+-dependent, cyclosporin A (CsA)-sensitive process due to the opening of a pore in the inner mitochondrial membrane leading to a collapse of ionic gradients and ultimately to mitochondrial dysfunction. We have recently shown that ammonia induced the MPT in cultured astrocytes. We intend to examine the role of the MPT in HE and hyperammonemia using ammonia-treated neural cell cultures and in vivo models of HE/hyperammonemia (HA). Our working hypothesis is that ammonia induces the MPT in astrocytes, culminating in mitochondrial failure and astroglial dysfunction. A corollary of this concept is that inhibition or interference in the development of the MPT in astrocytes may ameliorate CNS dysfunction in HE. The Specific Aims of this proposal are: 1) To identify the factors responsible for the ammonia-induced MPT in cultured neural cells. Our focus will be on agents implicated in the pathogenesis of HE/H that have also been shown to induce the MPT in other cells. Specifically, we will examine the role of Ca 2+, reactive oxygen species, nitric oxide, pH and glutamine. We will determine whether these factors are elevated in ammonia-treated cultures, and whether diminishing their production or blocking their actions reduces or abolishes the MPT. Additionally, we will examine possible sequential interrelationships among these factors. 2) To determine whether ammonia-induced abnormalities in astrocytes (morphological alterations, defects in neurotransmitter uptake, and cell swelling) are mediated by the MPT, we will investigate whether inhibitors of the MPT (CsA, bongkrekik acid) are capable of diminishing or blocking the deleterious effects of ammonia. 3) To investigate the involvement of mitochondrial dysfunction as a potential factor in MPT-mediated cell injury. We will determine the state of mitochondrial function after ammonia treatment, and then investigate whether improving energy metabolism will inhibit ammonia-induced cellular injury. 4) To clarify whether the MPT occurs in in vivo models of HE (thioacetarnide treatment) and hyperammonemia. We will also determine whether factors that inhibit the MPT in vitro (e.g., CsA, trifluoperazine) are also capable of doing so in vivo. Additionally, we will assess the ability of MPT blockers to improve the clinical, histopathologic, neurochemical abnormalities, and the extent of brain swelling observed in HE/HA. We believe that these studies will yield critical data bearing on the pathogenesis of HE, and may potentially aid in the development of novel therapeutic strategies for the treatment of this condition.